Human EWS-FLI protein levels and neomorphic functions show a complex, function-specific dose–response relationship in Drosophila

Ewing sarcoma (EwS) is a cancer that arises in the bones and soft tissues, typically driven by the Ewing’s sarcoma breakpoint region 1-Friend leukemia virus integration 1 (EWS-FLI) oncogene. Implementation of genetically modified animal models of EwS has proved difficult largely owing to EWS-FLI’s high toxicity. The EWS-FLI1FS frameshift variant that circumvents toxicity but is still able to perform key oncogenic functions provided the first study model in Drosophila. However, the quest for Drosophila lines expressing full-length, unmodified EWS-FLI remained open. Here, we show that EWS-FLI1FS’s lower toxicity is owed to reduced protein levels caused by its frameshifted C-terminal peptide, and report new strategies through which we have generated Drosophila lines that express full-length, unmodified EWS-FLI. Using these lines, we have found that the upregulation of transcription from GGAA-microsatellites (GGAAμSats) presents a positive linear correlation within a wide range of EWS-FLI protein concentrations. In contrast, rather counterintuitively, GGAAμSats-independent transcriptomic dysregulation presents relatively minor differences across the same range, suggesting that GGAAμSat-dependent and -independent transcriptional upregulation present different kinetics of response with regards to changing EWS-FLI protein concentration. Our results underpin the functional relevance of varying EWS-FLI expression levels and provide experimental tools to investigate, in Drosophila, the effect of the EWS-FLI ‘high’ and ‘low’ states that have been reported and are suspected to be important for EwS in humans.


Introduction
Ewing sarcoma (EwS) is an aggressive tumour, typically arising from bone and soft tissues, which is reported to be the second most common bone malignancy in children, adolescents and young adults [1][2][3].
EwS is a paradigm for solid tumour development caused by a single genetic lesion, a fusion between the transactivation domain of any member of the FET (FUS, EWSR1 and TAF15) family of RNA-binding proteins and the DNA-binding domain of one of several E26 transformation specific

The C-terminal peptide of the EWS-FLI 1FS variant results in decreased protein levels
The frameshift variant EWS-FLI 1FS was discovered as a serendipitous mutant that circumvented embryonic lethality, which was presumably caused by leaky expression from the Upstream Activating Sequence (UAS) of UAS>EWS-FLI (i.e.expression in the absence of any Gal4 driver).Functional assays showed that rather than to the loss of the 69 amino acid C-terminal peptide of EWS-FLI 1 , the lower toxicity of EWS-FLI 1FS was owing to a neomorphic property of the new FStail [17].We did not know the nature of such a neomorphic property, but a simple hypothesis was that it could be owing to reduced protein expression and/or stability.
To test this hypothesis, we quantified the effect of FStail tagging on YFP.To this end, we generated transgenic animals carrying UAS>YFP and UAS>YFP +FStail , a fusion of the FStail to the C-terminal end of YFP.By driving UAS>YFP and UAS>YFP +FStail expression from the nub>Gal4 driver, we found that both YFP +FStail green fluorescence intensity, as measured by confocal microscopy, as well as YFP +FStail protein levels, as quantified by western blot, are extremely reduced compared with those of unmodified YFP in salivary glands and wing imaginal discs (figure 1a,b).
Because the YFP and YFP +FStail transgenes were inserted at the same genomic site (attp40 [20]), by φC31 integrase-mediated transgenesis [21] and were both expressed by nub>Gal4, transcription levels are expected to be comparable.That is unless the FStail-coding sequence affects transcription, which could indeed explain the differences observed at the protein level.To test this hypothesis, we quantified YFP and YFP +FStail transcripts by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in salivary glands and wing imaginal discs.We found no significant differences in the level of mRNA from each variant in either tissue (figure 1c).Altogether, these results show that the FStail has no significant effect on transcription but affects protein expression and/or stability such that it results in a strong reduction in the concentration of a protein carrying this C-terminal tag.These results substantiate the hypothesis that EWS-FLI 1FS 's reduced toxicity is owing to a significant reduction in protein concentration caused by the FStail.
To further substantiate this conclusion, and to get a direct estimate of the effect of the FStail in EWS-FLI 1 , we generated the EWS-FLI 1+FStail transgene, an FStail-tagged version of full-length EWS-FLI 1 .We found that in stark contrast with unmodified EWS-FLI 1 , EWS-FLI 1+FStail transgenes were obtained at the standard rate of more than five per 200 injected embryos, thus confirming that the sole addition of the FStail can circumvent the toxicity issue of unmodified EWS-FLI 1 (figure 2).

Generating Drosophila strains carrying unmodified, full-length EWS-FLI transgenes
Having shown that EWS-FLI 1FS 's reduced toxicity is owing to a significant reduction in protein concentration caused by the FStail, we decided to test if the same effect could be achieved with transgenes designed to downregulate translation or transcription, so that full-length, untagged EWS-FLI 1 could be expressed.We used two approaches to this aim.
The first approach was to downregulate translation by placing an EWS-FLI 1 coding sequence lacking its own internal ribosome entry site as the secondary open reading frame (ORF) in a bicistronic transcript encoding mCherry as primary ORF.Under these conditions, translation of the secondary ORF is so severely compromised that it has been estimated to be essentially null in the absence of Gal4, hence avoiding potential toxicity issues caused by leaky transcription during transgenesis [22][23][24][25].
The second approach was based on P-element-mediated transgenesis [26,27].Unlike precise targeting to predetermined genomic sites by φC31 integrase [21], P-element transgenes insert nearly randomly in the genome and are, therefore, subjected to unpredictable position effects that may significantly affect transcription.We reasoned that this inherent drawback could be to our advantage because it may be used to select for insertions in genomic regions in which leaky UAS>EWS-FLI 1 expression is downregulated below toxic levels.
We found that both approaches work well to generate Drosophila lines carrying unmodified, full-length EWS-FLI 1 transgenes.φC31 integrase-mediated transgenic lines carrying the bicistronic construct bcEWS-FLI 1 were obtained at the standard rate of more than five per 200 injected embryos, thus showing that this strategy abrogates EWS-FLI 1 embryo toxicity.P-element-mediated transgenesis was much less efficient, producing a single pEWS-FLI transgenic line out of 180 injected embryos (<1%), which is one order of magnitude below the normal rate of P-element-mediated transgenesis (>10%) (figure 2).Such a low rate is not surprising, however, since only the small fraction of P-element insertions subjected to position effects strong enough to abrogate toxic, leaky EWS-FLI 1 expression can be expected to generate viable transgenic animals.DNA sequencing of the new transgenic lines obtained from both approaches confirmed the presence of non-mutated, full-length EWS-FLI 1 , as intended, thus validating the two strategies.

The collection of full-length EWS-FLI transgenes causes a range of developmental phenotypes
As a first step to characterize the new transgenic lines, we investigated their effect on development upon expression from a variety of Gal4 drivers (figure 3a).We found that as far as the lethality phase, wing disc tumours and cuticular phenotypes in adult flies are concerned, the new variants cause a wide range of expressivity levels.Top of the range is pEWS-FLI 1 that is lethal when expressed from any of the selected Gal4 drivers, including longGMR>Gal4 and sal EPv >Gal4 with which all of the other transgenes are viable.At the bottom of the range is the bcEWS-FLI 1 variant that yields pupal lethality with da>Gal4, Act5C>Gal4 and 48Y>Gal4, and adult viability with Mef2>Gal4 and twi>Gal4, while the same Gal4 drivers result in embryonic lethality with all other EWS-FLI 1 versions.Moreover, surviving adults carrying bcEWS-FLI 1 and Mef2>Gal4 or twi>Gal4 present no noticeable cuticular or lifespan phenotypes.EWS-FLI 1FS and EWS-FLI 1+FStail have an intermediate effect, stronger than bcEWS-FLI 1 and weaker than pEWS-FLI 1 (figure 3a).EWS-FLI 1FS and EWS-FLI 1+FStail are largely indistinguishable except for the effect on eye roughness and size when driven from sev>Gal4, which is stronger in the case of EWS-FLI 1+FStail (figure 3b, row 1).
To ameliorate the strong lethal effect of pEWS-FLI 1 , we also generated individuals carrying this construct together with a second transgene, P[tubP>GAL80 ts ], which expresses the temperature-sensitive allele of the yeast Gal80 protein from the alphatubulin84B promoter.Gal80 is an inhibitor of the transcriptional activation domain of Gal4.When expressed in Drosophila, Gal80 ts is fully functional at 17°C, but it is inactive at 29°C.By raising individuals carrying the pEWS-FLI 1 and P[tubP>GAL80 ts ] transgenes at 25°C we hoped to achieve a partial inactivation of Gal80 function that would in turn partially inhibit Gal4, hence lowering EWS-FLI 1 expression from UAS>pEWS-FLI 1 .We refer to this combination as pEWS-FLI 1 +Gal80@25.
The stronger effect of pEWS-FLI 1 compared with that of EWS-FLI 1FS and EWS-FLI 1+FStail is clearly reflected in their tumorigenic potential in wing imaginal discs.As published [17], EWS-FLI 1FS causes wing imaginal disc tumours when driven by nub>Gal4, but not by sal EPv >Gal4, which is expressed at a later stage in development and over a much smaller area in the wing disc.The same is true for EWS-FLI 1+FStail .In contrast, sal EPv >Gal4-driven expression is sufficient for pEWS-FLI 1 to cause wing imaginal disc tumours, and nub>Gal4-driven expression of pEWS-FLI 1 +Gal80@25 yields even larger tumours in the rare escapers (figure 3b, rows 2 and 3).

Upregulation of transcription from (GGAA)μSat and tissue toxicity present a positive linear correlation with a wide range of EWS-FLI protein concentrations
To further characterize the new full-length EWS-FLI 1 lines, we quantified the efficiency of each transgene at activating transcription from GGAAµSats, which is one of the hallmarks of the EWS-FLI 1 oncogene and is known to be critical for EwS tumorigenesis [9,18,28,29].The 20×(GGAA)µSat>YFP transgene was used to track GGAAµSat activation [17].We chose to carry out these studies on third instar larval salivary glands because they are among the tissues that are more resilient to the detrimental effects of EWS-FLI 1 [17].EWS-FLI 1 transgenes were expressed from sal EPv >Gal4 that, like other Gal4 drivers derived from the P{GawB} vector (Flybase FBtp0000352), contains a cryptic larval salivary-gland-specific enhancer element [30].
Using the same western blots, we re-assessed the levels of YFP expression from the 20×(GGAA)µSat>YFP brought about by each of the EWS-FLI 1 transgenes and found them to be largely consistent with those observed by confocal microscopy.This includes the fact that despite approximately 40% higher EWS-FLI 1 levels in pEWS-FLI 1 compared with pEWS-FLI 1 +Gal80@25 individuals, YFP expression levels are similar, hence suggesting that, as far as GGAAµSat transcriptional activation is concerned, EWS-FLI 1 levels are near saturation in pEWS-FLI 1 +Gal80@25 individuals.
Altogether, these results demonstrate a tight correlation of EWS-FLI protein levels to both their detrimental effect on salivary gland development and their effect on upregulating transcription from (GGAA)µSats.

GGAAμSat-independent transcriptome dysregulation is not linearly correlated with EWS-FLI protein levels
In spite of its frame-shifted C-terminal tail, EWS-FLI 1FS expression recapitulates in Drosophila key oncogenic functions of EWS-FLI 1 like transcriptional dysregulation of hundreds of genes including a fraction of the orthologues of known EWS-FLI's targets in human cells [17].The question, however, remains open as to whether unmodified full-length EWS-FLI 1 protein could bring about a transcriptomic signature that is closer to that of EwS tumours than the EWS-FLI 1FS 's signature.
To address this question, we used Affymetrix microarrays to perform a genome-wide transcriptomic analysis of salivary glands carrying the bcEWS-FLI 1 , and pEWS-FLI 1 +Gal80@25 transgenes that express very different levels of full-length EWS-FLI 1 (electronic supplementary material, table S1).EWS-FLI 1FS , salivary glands were also included as reference.For each condition, we determined the total number of dysregulated transcripts, quantified the enrichment of two published EwS signatures, and assessed the upregulation of neural genes, a feature that has been reported upon expression of EWS-FLI in human cells [31,32].
These are remarkable results as far as bcEWS-FLI 1 is concerned, taking into account the extremely low levels of EWS-FLI 1 protein and the lack of toxicity caused by this construct that nonetheless is nearly as good as EWS-FLI 1FS at driving GGAAµSatindependent transcriptome dysregulation.Altogether, these results show that, unlike GGAAµSat-dependent transcriptional upregulation, GGAAµSat-independent transcriptome dysregulation, including ectopic expression of orthologues of human EwS signature genes, is not linearly correlated with EWS-FLI 1 protein levels.

Discussion
EWS-FLI cytotoxicity has proven a great hindrance in the development of genetically tractable animal models [15].In Drosophila, a very clear distinction can be made between toxicity caused by UAS>EWS-FLI 1 in the absence of Gal4 and toxicity caused by Gal4-driven expression.The former, which accounts for the extreme difficulty in generating transgenic lines, can be circumvented by tagging the EWS-FLI 1 fusion with the FStail [17].The latter, however, is only partially reduced by the FStail.Here, we show that the FStail works by reducing protein levels, which in turn suggests that FStail tagging may facilitate the implementation of other animal models expressing EWS-FLI and perhaps other lethal oncogenic fusion proteins.
Building on this observation, we have generated new lines that express full-length, unmodified EWS-FLI 1 from transgenes that were engineered to reduce protein expression to a greater or lesser extent, hence circumventing the need for FStail tagging.
Using the new EWS-FLI 1 lines, it is possible to generate a phenotypic series, from which we have learned that the neomorphic effects brought about by EWS-FLI 1 that we have studied can be categorized in three classes on the basis of how they correlate with EWS-FLI 1 protein levels.The first includes the assembly of neo-enhancers at GGAAµSats that presents a positive linear correlation over a wide range of EWS-FLI 1 protein levels, from the lowest, (bcEWS-FLI 1 ) up to the highest (pEWS-FLI 1 +Gal80@25 and pEWS-FLI 1 ).The second includes massive GGAAµSat-independent transcriptional dysregulation including, notably, a significant fraction of orthologues of human EwS signature' genes.Rather counterintuitively, despite the order of magnitude lower level of expressed EWS-FLI 1 , the bcEWS-FLI 1 transgene is able to significantly dysregulate hundreds of genes albeit with lower fold changes.Finally, transcriptional upregulation of neural genes, a landmark of EWS-FLI activity in human cells, is only marginal in bcEWS-FLI 1 compared with EWS-FLI 1FS and pEWS-FLI+Gal80@25.These results reveal a complex, phenotype-specific correlation between EWS-FLI 1 levels and the expressivity of its phenotypes, that, in turn, implies fundamental differences in the mechanisms that drive these phenotypes.In particular, our results suggest that GGAAµSatdependent and GGAAµSat-independent transcriptional upregulation present different kinetics with regard to EWS-FLI protein concentration, opening a new perspective on EWS-FLI-dependent dysregulation of transcription.
royalsocietypublishing.org/journal/rsob Open Biol.14: 240043 Despite a low mutational burden characteristic of paediatric tumours, EwS displays high levels of intratumoral heterogeneity largely mediated by alterations to the epigenetic state.Several independent findings have suggested that differences in EWS-FLI levels are a determinant of different cellular phenotypes that may have direct implications for EwS progression [35][36][37][38][39][40][41][42][43].Altogether, our results highlight the functional relevance of EWS-FLI expression levels and provide experimental tools to further investigate in Drosophila the molecular pathways affected by the EWS-FLI 'high' and 'low' states observed in human tumours.

Immunohistochemistry and microscopy
Immunostaining of salivary glands and wing discs was performed as follows: salivary glands and wing discs were dissected in phosphate-buffered saline (PBS), fixed for 30 min in 4% formaldehyde with 0.3% TritonX-100, washed three times in PBS-0.3%Triton X-100 (PBST) for 10 min per wash.DNA was stained with DAPI.Salivary glands and wing discs were mounted in Vectashield (molecular probes).Immunostaining images were acquired with a Leica TCS SP8 scan unit coupled to a microscope and managed by Leica Application Suite X (LAS X) software.The objective used was HC PL APO CS2 40×/1.30OIL and images were acquired at zoom 1. Fluorophores 405 and 488 were excited with lasers diode and argon, respectively.Image processing was carried out with Fiji.All shown immunofluorescence images correspond to a single Z.

Quantification and statistical analysis
Quantification of YFP fluorescence levels was carried out using ImageJ to calculate the mean grey values of each region of interest (ROI) in a focal plane per salivary gland acquired with an SP8 Leica confocal image microscope.
Quantification of salivary gland size was performed in Inkscape 1.2.2 using Bezier curves to manually outline individual salivary glands from the distal end till the meeting point of the individual ducts at the proximal end and measure the area of the resulting shape through the visualized path extension.
Both salivary gland size and YFP intensity were represented in boxplots, p-values for salivary gland size and GGAAµSat activation were calculated in R version 4.

Cloning and transgenesis
The pUASt P-element EWS-FLI 1 construct was made by cloning the EcoRI-XhoI fragment from pUC-GW-Kan EWS-FLI 1 TI that contains the full EWS-FLI 1 ORF in the original pUASt vector [45].The pUAST-EWS-FLI 1+FStail construct was made by introducing a BbsI site into the pUC-GW-Kan EWS-FLI 1 TI.This was achieved by replacing the BsrGI-XhoI fragment with a gBlock (IDT) containing the BbsI site at the terminus of the ORF.Subsequently, another gBlock (IDT) with the FS sequence was inserted in-frame with EWS-FLI 1 into the BbsI and XhoI sites.The resulting fragment was subcloned into the EcoRI and XhoI sites of pUASt-attB (DGRC Stock 1419 [46]).The pUASt-uORF-EWS-FLI 1 construct was made by PCR amplification of the mCherry ORF from the pLT3-Dam vector [24] and the EWS-FLI 1 ORF from pUC-GW-Kan EWS-FLI 1 TI.Subsequently, both fragments were fused and cloned by in-fusion (Takara) into EcoRI and XhoI sites of the pUASt-attB vector.All PCR amplifications were performed using KOD polymerase (Merck).Cloning was performed by using In-Fusion Snap Assembly (Takara).Primers were ordered to Sigma.DNA fragments were ordered to IDT.
Transgenic fly lines were generated by BestGene Inc. (φC31 integrase-mediated transgenesis) using the Bloomington stock 24482, and by the Institute for Research in Biomedicine (IRB) Barcelona Drosophila Injection Service (P-element-mediated transgenesis) using the w 1118 line.

Microarray processing
Dissected Drosophila salivary glands were collected in 45 µl of a lysis buffer containing 20 mM dithiothreitol (DTT), 10 mM Tris-HCl pH 7.4, 0.5% sodium dodecyl sulfate (SDS) and 0.5 µg µl −1 proteinase K, incubated at 65°C for 15 min and immediately frozen until processing.RNA extraction and cDNA generation was performed at the IRB Barcelona Functional Genomics Core Facility.Briefly, RNA was treated with DNAse I and purified using magnetic beads (RNAClean XP, Beckman Coulter).RNA was quantitated with Qubit RNA HS Assay kit (Invitrogen), and RNA integrity was assessed with the Bioanalyzer 2100 RNA Pico assay (Agilent).Twenty-five nanograms of RNA were reverse transcribed and amplified using the whole transcriptome amplification method (WTA2, Sigma Aldrich) with 17 cycles of amplification.cDNA was further purified using a spin column (PureLink Quick PCR Purification Kit, Invitrogen) and quantified using a microvolume spectrophotometer (Nanodrop ND-1000, Thermo-Fisher Scientific).
For microarray processing, 8 µg of cDNA were fragmented and labelled according to the manufacturer's instructions (GeneChip Mapping 250K Nsp Assay Kit, Affymetrix).Array hybridization was performed using the GeneChip Hybridization, Wash and Stain Kit (Applied Biosystems).Briefly, libraries were denatured at 99°C for 2 min before incubation into the Drosophila Genome 2.0 arrays (Applied Biosystems).Libraries were hybridized on the arrays for 16 h at 45°C for 60 rpm at GeneChip Hybridization Oven 645 (Affymetrix/ThermoFisher Scientific).Washing and Stain steps were performed using a GeneChip Fluidics Station 450 following the Drosophila Genome 2.0 protocol (Affymetrix/ThermoFisher Scientific).Finally, arrays were scanned with a GeneChip Scanner GCS3000 (Affymetrix/ThermoFisher Scientific).The CEL files containing the microarray data were generated with the Command Console software (Affymetrix/ThermoFisher Scientific) and were used for probeset-based gene expression measurements using robust multichip average (RMA) normalization.Results were analyzed using the Transcriptome Analysis Console 4.0 (TAC) software.Genes with an absolute FC of >2 and an p<0.05 were considered differentially expressed.

Gene set enrichment analysis
The GSEA pre-ranked algorithm was used to compare the human EWS-FLI signatures from [33,34] to all genes in the Drosophila microarrays ranked by average log2FC.Genesets with an FDR q-value of <0.25 were accepted as a significant enrichment.Drosophila orthologues of these human signatures were identified using the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT) orthologue mapping online resource [47].

GO analysis
Functional annotation of GO terms was performed using the online tool database and annotation, Visualization and integrated discovery (DAVID) [48].GOTERM_BIOLOGICAL_PROCESS_4 terms with a p<0.05 were accepted as a significant enrichment.

Quantitative real-time PCR
Dissected Drosophila larval salivary glands were collected in 45 µl of a lysis buffer containing 20 mM DTT, 10 mM Tris-HCl pH 7.4, 0.5% SDS and 0.5 µg µl −1 proteinase K, incubated at 65°C for 15 min and immediately frozen until processing.RNA extraction and cDNA generation were performed at the IRB Barcelona Functional Genomics Core Facility.Briefly, RNA was treated with DNAse I and purified using magnetic beads (RNAClean XP, Beckman Coulter).RNA was quantitated with Qubit RNA HS Assay kit (Invitrogen), and integrity was assessed with the Bioanalyzer 2100 RNA Nano assay (Agilent).Twenty-five nanograms of RNA were reverse transcribed and amplified using the whole transcriptome amplification method (WTA2, Sigma Aldrich) with 17 cycles of amplification.cDNA was further purified using a spin column (PureLink Quick PCR Purification Kit, Invitrogen) and quantified using a microvolume spectrophotometer (Nanodrop ND-1000, Thermo-Fisher Scientific).cDNA yield ranged from 18 to 44 µg.PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) was used for quantitative real-time PCR following manufacturer's instructions.The real-time assays were conducted in a QuantStudio 6 Flex real-time PCR system (Thermo Fisher Scientific) using SYBR green as the detection system and ROX as the reference dye.Two different pairs of primers were designed to amplify YFP cDNA.mRNA levels were assessed from three independent RNA extractions and three technical replicates were performed on each sample.RNA levels were normalized to Ribosomal Protein L32 (RpL32) housekeeping gene.The primers used are shown in table 1.

Figure 3 .
Figure 3. Drosophila transgenic lines expressing full-length human EWS-FLI 1 generate a wide range of developmental phenotypes.(a) Summary of developmental phenotypes observed in each EWS-FLI 1 transgenic line, colour coded with red for embryonic lethal, yellow for larval or pupal lethal and cyan for adult viable.(b) Adult eye and third instar wing imaginal discs from individuals expressing the indicated versions of EWS-FLI 1 in ommatidia (sev-Gal4), central region of the wing pouch (sal EPv >Gal4) or wing pouch and hinge region (nub>Gal4).The nub>Gal4/UAS>EWS-FLI 1FS and nub>Gal4/UAS>EWS-FLI 1+FStail wing disc tumours shown are extreme examples to demonstrate the extent of transformation that can be induced by these transgenes; milder phenotypes have been observed.Most nub>Gal4/UAS>PEWS-FLI 1 ; P[tubP>GAL80 ts ]/+ individuals reared at 25°C die at early stages of development; only two third instar larval escapers that we have observed presented large wing disc tumours (+Gal80 ts 25°C).Scale bars: 0.1 mm or 100 µm for adult eyes and wing discs, respectively.
2.3.by Dunn's test with Holm's method for p-value adjustment after rejection of Kruskal-Wallis one-way ANOVA and compact letter display (CLD) was used to represent p-values on boxplots.Values of p for western blot band intensities were calculated by Tukey's HSD after the rejection of one-way ANOVA.

Table 1 .
Primers used in this article.